The importance of protein bioconjugates has grown as they have found widespread use as therapeutics, chemical sensors, scaffolds for new materials, and tools for basic research. The construction of such materials depends on the ability to modify proteins selectively and quantitatively, producing a continual demand for new strategies. While myriad methods for protein modification exist, it remains difficult to modify native proteins in a well-defined manner. This problem is particularly apparent with one of the most common methods for protein modification: acylation of lysine residues with NHS-esters. This modification strategy results in inseparable statistical mixtures of proteins with differing levels of modification. It is possible to circumvent this limitation by using chemistries that target rare (engineered cysteines, tyrosines), unique (N-terminus, C-terminus), or introduced (small molecule tags, peptide tags) moieties on proteins, but the applicability of these methods varies on a case-by-case basis.
This problem has motivated much of current research into strategies for site-selective protein modification, and it has slowed the development of many protein-based materials. For example, it is known that the drug loading on antibody-drug conjugates affects their efficacy, yet traditional methods like lysine acylation often result in over-modification. It remains challenging to construct antibody-drug conjugates with controlled levels and locations of drug loading. This problem has been encountered during the construction of mimics of light harvesting arrays. Efficient light-harvesting systems require the precise arrangement of multiple unique dyes. These systems have been successfully approximated by using viral coat proteins to template pairs of dyes that participate in Förster resonance energy transfer (FRET). However, because the modification site on each protein monomer is chemically identical, it is impossible to control the exact ratio of acceptors to donors on each protein assembly in a precise manner. As a result, these efforts have been limited to the study of statistical mixtures of light-harvesting mimics with different dye compositions.
While the development of site-selective bioconjugation reactions certainly has a role to play in addressing these problems, one missing piece is a general separation technique that can distinguish between bioconjugates with different levels and sites of modification. Such a technique would be akin to silica gel chromatography, which is used frequently for small molecule synthesis.
Those who modify proteins seldom use chromatography to separate proteins based on their degree of modification because most available methods-including gel filtration, ion-exchange chromatography, and hydrophobic interaction chromatography-poorly discern the small differences in polarity, charge, and size brought about by modification with small molecules. In contrast, affinity chromatography has the unique ability to select for proteins bearing small, specific chemical groups. This technique has been used in specific instances to purify proteins modified with certain groups like fluorescent dyes or prenyl groups. However, it has not been explored as a general method for the isolation of homogeneous bioconjugates that are modified with arbitrary chemical moieties. There is a need for general methods for separation of proteins based on degree of modification and improved methods for preparation of well-defined bioconjugates. The present invention meets these and other needs.